Methods of immunizing adults using anti-meningococcal vaccine compositions

ABSTRACT

A method for boosting an immune response against meningococcal capsular antigen is disclosed. The method entails administering a first glycoconjugate vaccine composition to a subject to provide an initial state of anti-meningococcal immunity, and then boosting the anti-meningococcal immunity by administration of a second, boosting vaccination. Also disclosed is the use of vaccine compositions in the preparation of anti-meningococcal medicaments. The use entails administering a first glycoconjugate vaccine composition to a subject to provide an initial state of anti-meningococcal immunity, and then boosting the anti-meningococcal immunity by administration of a second, boosting vaccination.

CROSS-REFERENCE TO RELATED APPLICATION

This application is related to provisional patent application Ser. No.60/050,581, filed Jun. 24, 1997, from which priority is claimed under 35U.S.C. §119 (e) (1) and which is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates to methods for immunizing a subjectagainst meningococcal disease. More particularly, the invention relatesto a method for avoiding immunological tolerance against meningococcalspecies in vaccinated subjects, using an anti-meningococcalglycoconjugate vaccine composition as the primary immunogen.

BACKGROUND OF THE INVENTION

Neisseria meningitidis is an important cause of invasive bacterialdisease, with an estimated 2600 cases of meningococcal meningitisoccurring in the United States each year, primarily in children andyoung adults (Jafari et al. (1997) MMWR 46:1-10; Perkins et al. (1997)MMWR 46:13-21). In England and Wales, there has been a steady rise inreported cases of meningococcal disease since 1984, peaking at greaterthan 2000 cases per year in 1991 and 1992 (Jones, D. (1995)“Epidemiology of Meningococcal Disease in Europe and the USA, inMeningococcal disease, Cartwright, K. (ed), John Wiley & Sons Ltd., pp.147-157). Despite antimicrobial therapy, mortality rates in Europe andin the USA from meningococcal disease remain high (i.e., 12 to 14percent of cases, Jafari, supra and Jones, supra.) In mostindustrialized countries, the vast majority of isolates causingmeningococcal disease are serogroups B or C (Jones, supra; Harrison, L.(1995) JAMA 273:419-421; Jackson et al. (1995) JAMA 273:383-389; Whalenet al. (1995) JAMA 273:390-394). For reasons that are unknown, serogroupA strains, the major cause of disease in developing countries (Harrison,supra) are very rare in industrialized countries.

A substantial portion of meningococcal disease is potentiallypreventable by vaccination (Artenstein et al. (1970) N. Engl. J. Med.282:417-420; Reingold et al. (1985) Lancet 2:114-118; Peltola et al.(1977) N. Engl. J. Med. 297:686-691). Effective polysaccharide vaccinesagainst disease caused by serogroup A and C strains have been availablefor more than 20 years and, more recently, tetravalent vaccines havebeen licensed for prevention of serogroups A, C, Y and W135 isolates(Armand et al. (1982) J. Biol. Stand. 10:335-339; Arnbrosch et al.(1983) Bull. World Health Organ. 61:317-323).

Despite their general availability, meningococcal polysaccharidevaccines are used infrequently in industrialized societies (Harrison,supra). For example, in the United States, vaccination is largelylimited to certain high risk situations, such as with patients withfunctional asplenia or terminal complement deficiency diseases (Jafari,supra). Vaccination is also used for controlling meningococcal diseasein military recruits (Harrison, supra), and may be beneficial forhealthy individuals traveling to hyperendemic areas, and for control ofcivilian outbreaks of meningococcal disease caused by serogroup strainsincluded in the available vaccines (Perkins, supra; Masterton et al.(1988) J. Infect. 17:177-182). The reasons for the limited use ofmeningococcal polysaccharide vaccines in the general population includetheir poor immunogenicity in infants less than 2 years of age, the agegroup at greatest risk of developing meningococcal disease (Jafari,supra; Jones, supra) In addition, the duration of vaccine-inducedprotection elicited in older children and adults is limited (Zangwill etal. (1994) J. Infect. Dis. 169:847-852). Finally, these polysaccharidevaccines provide no protection against disease caused by serogroup Bstrains, which accounts for approximately 40% of all cases in the UnitedStates (Jafari, supra) and Canada (Whalen, supra), and an even greaterproportion in the United Kingdom (Jones, supra).

More effective polysaccharide-protein conjugate vaccines for preventionof disease caused by meningococcal A and C strains are currently underdevelopment (reviewed in Granoff et al. (1997) Int. J. Infect. Dis.1:152-157). These conjugate vaccines are immunogenic in infants andtoddlers, and elicit high titers of serum bactericidal antibody (Fairley(1996) J. Infect. Dis. 174:1360-1363, and Lieberman et al. (1996) JAMA275:1499-1503).

In addition, polysaccharide derivatives have been prepared to circumventdisease caused by meningococcal B strains. For example, C₄-C₈N-acyl-substituted polysaccharide derivatives have been described. See,EP Publication No. 504,202 B, to Jennings et al. Similarly, U.S. Pat.No. 4,727,136 to Jennings et al. describes an N-propionylatedmeningococcal B polysaccharide molecule. Mice immunized withglycoconjugates formed with these polysaccharide derivatives werereported to elicit high titers of IgG antibodies. Jennings et al. (1986)J. Immunol. 137:1708. More recently, meningococcal B oligosaccharidederivative fragments, and glycoconjugates made from those fragments,have been shown to be highly effective immunogens for use inanti-meningococcal B vaccine preparations. International Publication No.WO 98/086543.

Although anti-meningococcal conjugate vaccine preparations are moreeffective than unconjugated vaccines in infants and toddlers,unconjugated polysaccharide vaccines are highly immunogenic in adults,eliciting effective short-term protection against disease (Artenstein,supra., Gold et al. (1971) Bull. World Health Organ. 45:279-282). Thus,there appears to be no real advantage to using the more costlymeningococcal conjugate vaccine in adults. Indeed, in comparativeimmunogenicity studies, the magnitude of the serum antibody response ofadults given a dose of unconjugated pneumococcal or meningococcalpolysaccharide vaccine appears to be similar to that elicited by thecorresponding polysaccharide-protein conjugate vaccine (Anderson et al.(1994) Infect. Immun. 62:3391-3395; Powers et al. (1996) J. Infect. Dis.173:1014-1018).

SUMMARY OF THE INVENTION

It is a primary object of the invention to provide a method for boostingin an adult subject an immune response against meningococcal capsularantigen. The method generally entails the steps of (a) administering afirst vaccine composition to an adult subject in order to elicit animmune response against a meningococcal species, and (b) administering asecond vaccine composition to said adult subject in order to boost theanti-meningococcal response. The first vaccine composition comprises ameningococcal oligosaccharide conjugated to a carrier molecule, whereinthe composition is administered in an amount sufficient to elicit ananti-meningococcal immune response, and said immune response isboostable upon revaccination with a second meningococcal vaccinecomposition. The second vaccine composition is administered to thesubject after serum anti-meningococcal antibody concentration induced bythe first vaccine composition have declined to subprotective levels.

It is also an object of the invention to provide a use of a first andsecond meningococcal polysaccharide or oligosaccharide composition inthe preparation of a medicament. The first composition comprises ameningococcal oligosaccharide conjugated to a carrier molecule, and isadministered in an amount sufficient to elicit an anti-meningococcalimmune response which is boostable upon re-vaccination with a secondmeningococcal vaccine composition. The second composition comprises ameningococcal polysaccharide or oligosaccharide immunogen, and isadministered to the subject after serum anti-meningococcal antibodyconcentrations induced by the first vaccine composition have declined tosub-protective levels.

It is an advantage of the present invention that the methods and usescan be employed in an anti-meningococcal vaccination protocol whichavoids problems associated with induction of immunological tolerance tomeningococcal immunogens as seen with prior vaccination strategies.

It is also a feature of the present invention that a wide variety ofcommonly available anti-meningococcal capsular oligosaccharide orpolysaccharide glycoconjugates may be used as the immunogen in the firstvaccine composition.

Additional objects, advantages and novel features of the invention willbe set forth in part in the description which follows, and in part willbecome apparent to those skilled in the art upon examination of thefollowing, or may be learned by practice of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the geometric mean antibody response to a booster dose ofthe quadravalent meningococcal A, C, Y, W135 polysaccharide vaccine(MENOMUNE™) in naive subjects, and in subjects previously given either(a) a full dose of the tetravalent meningococcal polysaccharide vaccine(MENOMUNE™), or (b) a dose of an investigational meningococcal A and Coligosaccharide-protein conjugate vaccine.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of immunology, microbiology, molecularbiology and recombinant DNA techniques within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., Sambrook,et al. Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); DNACloning: A Practical Approach, vol. I & II (D. Glover, ed.);Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic AcidHybridization (B. Hames & S. Higgins, eds., 1985); Transcription andTranslation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R.Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning(1984); and Handbook of Experimental Immunology, Vols. I-IV (D. M. Weirand C. C. Blackwell eds., 1986, Blackwell Scientific Publications).

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural references unless the contentclearly dictates otherwise.

I. Definitions

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

An “antigen” is defined herein to include any substance that may bebound by an antibody molecule. An “immunogen” is an antigen that iscapable of initiating lymphocyte activation resulting in anantigen-specific immune response. Such activation generally results inthe development of a secretory, cellular and/or antibody-mediated immuneresponse against the immunogen. Usually, such a response includes but isnot limited to one or more of the following effects; the production ofantibodies from any of the immunological classes, such as IgA, IgD, IgE,IgG or IgM; the proliferation of B and T lymphocytes; the provision ofactivation, growth and differentiation signals to immunological cells;expansion of helper T cell, suppressor T cell, and/or cytotoxic T celland/or γδ T cell populations. Immunogens therefore include any moleculewhich contains one or more antigenic determinants (e.g., epitopes) thatwill stimulate a host's immune system to initiate such anantigen-specific response.

By “epitope” is meant a site on an antigen to which specific B cells andT cells respond. The term is also used interchangeably with “antigenicdeterminant” or “antigenic determinant site.” A peptide epitope cancomprise 3 or more amino acids in a spatial conformation unique to theepitope. Generally, an epitope consists of at least 5 such amino acidsand, more usually, consists of at least 8-10 such amino acids. Methodsof determining spatial conformation of amino acids are known in the artand include, for example, x-ray crystallography and 2-dimensionalnuclear magnetic resonance. Furthermore, the identification of epitopesin a given protein is readily accomplished using techniques well knownin the art. See, e.g., Geysen et al. (1984) Proc. Natl. Acad. Sci. USA81:3998 (general method of rapidly synthesizing peptides to determinethe location of immunogenic epitopes in a given antigen); U.S. Pat. No.4,708,871 (procedures for identifying and chemically synthesizingepitopes of antigens); and Geysen et al. (1986) Molecular Immunology23:709-715 (technique for identifying peptides with high affinity for agiven antibody). Antibodies that recognize the same epitope can beidentified in a simple immunoassay showing the ability of one antibodyto block the binding of another antibody to a target antigen.

As used herein, “treatment” refers to any of (i) prevention of infectionor reinfection, as in a traditional vaccine, (ii) reduction orelimination of symptoms, and (iii) reduction or complete elimination ofthe pathogen in question. Treatment may be effected prophylactically(prior to infection) or therapeutically (following infection).

By “mammalian subject” is meant any member of the class Mammalia,including, without limitation, humans and other primates, including suchnon-human primates as chimpanzees and other apes and monkey species;farm animals such as cattle, sheep, pigs, goats and horses; domesticmammals such as dogs and cats; and laboratory animals including rodentssuch as mice, rats and guinea pigs. The term does not denote aparticular age or sex. Thus, both adult and newborn individuals, as wellas fetuses, either male or female, are intended to be covered.

II. Modes of Carrying Out the Invention

The present invention is premised, in part, on the unexpected discoverythat use of an anti-meningococcal conjugate vaccine composition inadults (instead of an unconjugated anti-meningococcal polysaccharidevaccine) induces polysaccharide-responsive memory B cells and long-termimmunologic memory in vaccinated subjects, both of which factorscontribute to more robust and durable protection against meningococcaldisease. In fact, it has surprisingly been found that theanti-meningococcal immune response in adults vaccinated with a conjugatevaccine formulation is readily boostable upon re-vaccination with asecond anti-meningococcal vaccine composition.

In contrast, it has also been found herein that vaccination with anunconjugated tetravalent meningococcal A, C, Y, W135 polysaccharidevaccine (MENOMUNE™, Connaught Laboratories, Inc., Swiftwater, Pa.)induces immunologic paralysis of toddlers and adults to meningococcalpolysaccharides. More particularly, meningococcal C vaccination with apolysaccharide vaccine (unconjugated) in subjects during the first sixmonths of age results in depression of serum antibody responses to abooster vaccination with meningococcal C polysaccharide given 6 monthslater (when compared to the responses of infants of similar agevaccinated for the first time). In the study described hereinbelow,infants were given two doses of a meningococcal A and C polysaccharidevaccine at 3 and 6 months of age and boosted with a third injection at18 to 24 months of age. As shown in FIG. 1, the geometric mean antibodyresponse to the booster dose was nearly 10-fold lower than that ofcontrol children of the same age vaccinated for the first time. This newinformation on induction of immunologic tolerance in these vaccinatedsubjects indicates that such tolerance is not limited to infants lessthan 6 months of age, but also occurs in toddlers vaccinated at 15 to 23months, and in adults. In fact, antibody refractoriness in adultsubjects was observed 4 years after a polysaccharide vaccination.

The induction of immunologic tolerance to meningococcal species inpreviously vaccinated subjects is of significant clinical importance. Inthis regard, data from experimental animals indicate that mice tolerizedto pneumococcal polysaccharide show increased lethality fromexperimental challenge with pneumococci possessing the homologousserotype (reviewed in Halliday, W. (1971) Bacteriol. Rev. 35:267-289).This increased susceptibility may be a result of an impaired ability tomount serum anticapsular antibody responses upon exposure to theencapsulated bacteria. In humans, the contemporary knowledge acceptsthat unconjugated meningococcal polysaccharide vaccine are protective inadults and, possibly, in older children. However, this knowledge isgenerally based upon efficacy data in adults that were obtained fromstudies performed in military recruits in which follow-up was very short(8 weeks). As has been discovered herein, there may be a late-onsetincreased risk of disease in vaccinated subjects as a result of immunerefractoriness to meningococcal polysaccharides, once increased serumantibody concentrations induced by vaccination have declined tosub-protective levels (i.e., after about 3 years). The impairedmeningococcal C serum bactericidal antibody responses of toddlers andadults previously vaccinated with the tetravalent polysaccharide vaccineis consistent with this possibility.

Taken together, the data presented herein raise a safety concern for theuse of unconjugated anti-meningococcal vaccines, as this product isrecommended in the United States for use in children two years of age orolder, but the vaccine also can be used in infants and younger childrento control outbreaks of disease (Jafari, supra and Perkins, supra).

Accordingly, it is a primary object of the invention to provide a methodfor boosting in an adult subject an anti-meningococcal immune responseagainst a meningococcal capsular polysaccharide antigen. The methodgenerally entails a primary vaccination using a anti-meningococcalglycoconjugate vaccine composition which comprises meningococcalcapsular polysaccharide antigen derived from one or more meningococcalspecies (i.e., a monovalent or polyvalent vaccine composition)conjugated to an appropriate carrier molecule. The primary vaccinationis sufficient to elicit an anti-meningococcal immune response in thevaccinated subject which is specific for one or more meningococcalspecies. After the immune response elicited by the primary vaccinationhas declined to subprotective levels, a boosting vaccination isperformed in order to provide a boosted anti-meningococcal immuneresponse.

The anti-meningococcal glycoconjugates used for the primary vaccinationare prepared using carrier molecules that will not themselves induce theproduction of harmful antibodies. Suitable carriers are typically large,slowly metabolized macromolecules such as proteins, polysaccharides,polylactic acids, polyglycolic acids, polymeric amino acids, amino acidcopolymers, lipid aggregates (such as oil droplets or liposomes), andinactive virus particles. Preferably, capsular meningococcalpolysaccharide or oligosaccharide molecules containing at least oneimmunologically relevant epitope are conjugated to a bacterial toxoidcarrier molecule, such as, but not limited to, a toxoid from diphtheria,tetanus, cholera, etc. In particular embodiments, capsularpolysaccharide molecules are coupled to the CRM₁₉₇ protein carrier. TheCRM₁₉₇ carrier is a well-characterized non-toxic diphtheria toxin mutantthat is useful in glycoconjugate vaccine preparations intended for humanuse. (Bixler et al. (1989) Adv. Exp. Med. Biol. 251:175, and Constantinoet al. (1992) Vaccine). In other embodiments, glycoconjugates are formedwith protein carriers known to have potent T-cell epitopes. Exemplarycarriers include, but are not limited to, Fragment C of tetanus toxin(TT), and the Class 1 or Class 2/3 OMPs of N. meningitidis. Suchcarriers are well known to those of ordinary skill in the art.

In particular embodiments of the invention, the primary vaccinationentails administration of a meningococcal A and C oligosaccharide-basedglycoconjugate vaccine composition as described by Anderson et al.(supra). In other related embodiments, the primary vaccination is givenusing a meningococcal B oligosaccharide-based glycoconjugate asdescribed in International Publication No. WO 98/086543, whichpublication is incorporated herein by reference in its entirety. Othervaccine compositions that can be used herein for the primary vaccinationinclude, for example, glycoconjugates based on meningococcal Bpolysaccharide derivatives (e.g., those described in EP Publication No.504,202 B and U.S. Pat. No. 4,727,1361 both of which are incorporatedherein by reference), and monovalent meningococcal C or trivalentmeningococcal A, B and C oligosaccharide-based glycoconjugates.

The secondary (boosting) vaccination can be carried out using anysuitable anti-meningococcal vaccine composition; however, the secondvaccine composition is preferably also a meningococcal capsularpolysaccharide- or oligosaccharide-based conjugate in order to avoid thepossibility of immunologic tolerance associated with unconjugatedanti-meningococcal vaccine compositions.

As will be known by those skilled in the art upon reading the instantspecification, several factors will have an impact on the physical andimmunological properties of the above-described glycoconjugates.Specifically, the ratio of oligosaccharide (and/orpolysaccharide)-to-protein (hapten loading density), linkage chemistry,and the choice of carrier moiety are all factors that should beconsidered and optimized in the preparation of the glycoconjugates usedin the methods herein. For example, a low saccharide loading density mayresult in poor anti-saccharide antibody response. On the other hand, aheavy loading of saccharides could potentially mask important T-cellepitopes of the protein molecule, thus abrogating the carrier effect andattenuating the total anti-saccharide immune response.

Accordingly, during glycoconjugate production, aliquots can be withdrawnand analyzed by SEC-HPLC in order to monitor the extent of theconjugation process. The use of a disaggregating buffer, for exampleEDTA, SDS, deoxycholate, or the like, can be employed to separatecomponents possibly adhering to the preparations by non-covalentinteractions. To ensure glycosylation of the carrier, the shift inretention time of the particular protein carrier toward the exclusionvolume (V₀) of the column can be monitored. In addition, a gradualreduction of the saccharide peak area in a HPLC chromatogram can be usedto indicate incorporation of the saccharide onto the carrier.

Post-production characterization of the glycoconjugates can includemolecular weight determination using, for example, gel filtrationcolumns. Further characterization may also include electrophoreticmobility on SDS-PAGE separation equipment and analysis of chemicalcomposition of the glycoconjugates with respect to carbohydrate andamino acid components. The identity of product purity, and the absenceof residual contaminants (such as nucleic acids, LPS, and freesaccharides and/or carrier) can also be verified using known techniques.Confirmation of stable covalent attachment can be accomplished using acombination of analytical techniques, including gel filtration indetergent-containing buffer, SDS-PAGE followed by Western Blot analysisand amino acid analysis. See, e.g., Vella et al. (1992) Vaccines: NewApproaches to Immunological Problems, (Ellis, R. W. ed),Butterworth-Heinemann, Boston, pp 1-22, Seid et al. (1989)Glycoconjugate J. 6:489.

The anti-meningococcal vaccine compositions used in the primary andsubsequent (boosting) vaccinations can further be administered inconjunction with other antigens and immunoregulatory agents, forexample, immunoglobulins, cytokines, lymphokines, and chemokines,including but not limited to IL-2, modified IL-2 (cys125→ser125),GM-CSF, IL-12, γ-interferon, IP-10, MIP1β and RANTES.

The vaccine compositions will generally include one or more“pharmaceutically acceptable excipients or vehicles” such as water,saline, glycerol, ethanol, etc. Additionally, auxiliary substances, suchas wetting or emulsifying agents, pH buffering substances, and the like,may be present in such vehicles.

Adjuvants may also be used to enhance the effectiveness of the vaccines.Adjuvants can be added directly to the vaccine compositions or can beadministered separately, either concurrently with or shortly after,administration of the vaccines. Such adjuvants include, but are notlimited to: (1) aluminum salts (alum), such as aluminum hydroxide,aluminum phosphate, aluminum sulfate, etc.; (2) oil-in-water emulsionformulations (with or without other specific immunostimulating agentssuch as muramyl peptides (see below) or bacterial cell wall components),such as for example (a) MF59 (International Publication No. WO90/14837), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85(optionally containing various amounts of MTP-PE (see below), althoughnot required) formulated into submicron particles using a microfluidizersuch as Model 110Y microfluidizer (Microfluidics, Newton, Mass.), (b)SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymerL121, and thr-MDP (see below) either microfluidized into a submicronemulsion or vortexed to generate a larger particle size emulsion, and(c) RIBI™ adjuvant system (RAS), (Ribi Immunochem, Hamilton, Mont.)containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cellwall components from the group consisting of monophosphorylipid A (MPL),trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferablyMPL+CWS (DETOX™); (3) saponin adjuvants, such as STIMULON™ (CambridgeBioscience, Worcester, Mass.) may be used or particle generatedtherefrom such as ISCOMs (immunostimulating complexes); (4) CompleteFreunds Adjuvant (CFA) and Incomplete Freunds Adjuvant (IFA); (5)cytokines, such as interleukins (IL-1, IL-2, etc.), macrophage colonystimulating factor (M-CSF), tumor necrosis factor (TNF), etc.; (6)detoxified mutants of a bacterial ADP-ribosylating toxin such as acholera toxin (CT), a pertussis toxin (PT), or an E. coli heat-labiletoxin (LT), particularly LT-K63 (where lysine is substituted for thewild-type amino acid at position 63) LT-R72 (where arginine issubstituted for the wild-type amino acid at position 72), CT-S109 (whereserine is substituted for the wild-type amino acid at position 109), andPT-K9/G129 (where lysine is substituted for the wild-type amino acid atposition 9 and glycine substituted at position 129) (see, e.g.,International Publication Nos. W093/13202 and W092/19265); and (7) othersubstances that act as immunostimulating agents to enhance theeffectiveness of the composition.

Muramyl peptides include, but are not limited to,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acteyl-normuramyl-L-alanyl-D-isogluatme (nor-MDP),N-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-huydroxyphosphoryloxy)-ethylamine(MTP-PE), etc.

Typically, the vaccine compositions are prepared as injectables, eitheras liquid solutions or suspensions; or as solid forms suitable forsolution in, or suspension in, liquid vehicles prior to injection. Thepreparation also may be emulsified or encapsulated in liposomes forenhanced adjuvant effect.

The vaccine compositions will comprise a therapeutically effectiveamount of one or more meningococcal capsular oligosaccharide orpolysaccharide immunogens, and any other of the above-mentionedcomponents, as needed. By “therapeutically effective amount” is meant anamount of a molecule which will induce an immunological response in theindividual to which it is administered without stimulating an autoimmuneresponse. Such a response will generally result in the development inthe subject of a secretory, cellular and/or antibody-mediated immuneresponse to the vaccine. Usually, such a response includes but is notlimited to one or more of the following effects; the production ofantibodies from any of the immunological classes, such asimmunoglobulins A, D, E, G or M; the proliferation of B and Tlymphocytes; the provision of activation, growth and differentiationsignals to immunological cells; expansion of helper T cell, suppressor Tcell, and/or cytotoxic T cell and/or γδ T cell populations.

Preferably, the effective amount is sufficient to bring about treatment,i.e., reduction or complete elimination of symptoms, or prevention ofdisease symptoms. The exact amount necessary will vary depending on thesubject being treated; the age and general condition of the subject tobe treated; the capacity of the subject's immune system to synthesizeantibodies; the degree of protection desired; the severity of thecondition being treated; the particular molecule selected and its modeof administration, among other factors. An appropriate effective amountcan be readily determined by one of skill in the art. A “therapeuticallyeffective amount” will fall in a relatively broad range that can bedetermined through routine trials. More particularly, the meningococcalcapsular oligosaccharide or polysaccharide immunogens will beadministered in a therapeutically effective amount that comprises fromabout 0.1 μg to about 100 mg, more preferably from about 0.5 μg to about1 mg, and most preferably about 1 μg to about 500 μg of theoligosaccharide or polysaccharide immunogen delivered per dose.

Once formulated, the vaccine compositions are conventionallyadministered parenterally, e.g., by injection, either subcutaneously orintramuscularly. Alternative formulations suitable for other modes ofadministration include oral and pulmonary formulations, suppositories,and transdermal applications. Dosage treatment may be a single doseschedule or a multiple dose schedule.

III. Experimental

The following studies were designed to assess whether meningococcalconjugate vaccination of adults induces immunologic memory tounconjugated meningococcal C polysaccharide. To address this question,adults who had been immunized three to four years earlier with aninvestigational meningococcal A and C conjugate vaccine werere-vaccinated with unconjugated tetravalent meningococcal polysaccharidevaccine. The serum antibody responses to this booster injection werecompared to those of previously unvaccinated adults, or those of adultspreviously vaccinated with unconjugated meningococcal polysaccharidevaccine. Since “unprimed” adults were expected to show very high serumantibody responses to unconjugated meningococcal polysaccharide vaccine,the dose of meningococcal polysaccharide vaccine that was used for thebooster injection was chosen such that it would normally be consideredsuboptimally immunogenic (1/50 of the usual dose, to serve as a probe ofB cell immunologic memory).

A. Methods

Subjects: The following study was approved by the Saint Louis UniversityInstitutional Review Board. Thirty-four healthy adults, ages 20 to 53years, were divided into three groups based on their previousmeningococcal vaccination histories. Group 1 consisted of 5 subjects,each of whom had received a full dose of a U.S. licensed tetravalentmeningococcal polysaccharide vaccine (MENOMUNE™, 50 μg of A, C, Y, andW135 polysaccharides per 0.5 ml dose). This dose was given four yearsearlier as part of a previous study (Anderson, supra). Group 2 consistedof 18 subjects who had received a dose of an investigationalmeningococcal A and C oligosaccharide-protein conjugate. Fifteen ofthese subjects had been immunized four years earlier as part of the samestudy (Anderson, supra). The remaining three subjects were vaccinatedthree years earlier in a subsequent trial (Anderson et al., unpublisheddata).

The conjugate vaccine used in the studies contained 22 μg each of GroupA and C oligosaccharides and 48.7 μg of CRM₁₉₇ protein (a cross-reactivemutant nontoxic diphtheria toxin). Four of the subjects in group 2received the full 22 μg dose, 11 received an 11 μg dose (including thethree subjects vaccinated in the second trial), and 3 received a 5.5 μgdose. All doses were adsorbed to 1 mg of aluminum hydroxide and given ina 1 ml dose. In the present study, the magnitude and kinetics of thebooster antibody responses of the subjects previously given differentdoses of conjugate vaccine were very similar. Therefore, forpresentation of the results, the data from the three priming doses werecombined. Group 3 consisted of 11 adults who had not previously receivedmeningococcal vaccine. Two of the subjects in this group had beenrandomized in the first study to receive a saline placebo injection(Anderson, supra), and the remaining 9 subjects were previouslyunvaccinated healthy adults recruited as controls for the presentbooster study.

The demographic characteristics of the three “priming” vaccine groupswere similar with respect to median age at the time of the boostervaccination (38, 36, and 40 years of age for groups 1, 2, and 3,respectively), gender (predominantly female: 100%, 89%, and 82%,respectively), and race distribution (white: 100%, 94%, and 91%,respectively).

Vaccinations: After informed consent, all 34 healthy adults in groups 1,2 and 3 were vaccinated with {fraction (1/50)} of the usual dose of aquadravalent meningococcal A, C, Y, and W135 polysaccharide vaccine (1ml containing 1 μg of each polysaccharide, given IM in the deltoid). Toprepare this dose, lyophilized meningococcal polysaccharide vaccine(MENOMUNE™) from Connaught Laboratories (Swiftwater, Pa., U.S.) wasreconstituted with 0.6 ml of diluent provided by the manufacturer. Theresulting solution contained 100 μg/ml of each of the polysaccharides.From this solution, 0.5 ml was diluted into 49.5 ml of preservative-freesaline for injection, to yield the 1 ml dose. Serum samples wereobtained immediately prior to vaccination (time 0), and 3, 7 and 28 dayslater, for measurement of antibody response to the meningococcal Ccomponent of the vaccine.

Immunoassays: All assays were performed “blindly” on coded serumsamples. Serum anti-N meningitidis group C polysaccharide antibodyconcentrations were measured by an enzyme-linked immunosorbent assay(ELISA), adapted from a method previously described (Granoff et al.(1997) Infect. Immun. 65:1710-1715). In the present study, the assayemployed an alkaline-phosphatase conjugated mouse monoclonal antibodyspecific for human IgG (clone HP6083) (Granoff et al. (1995) Clin.Diagn. Lab. Immunol. 1:574-582). Also, the buffer for diluting the serumsamples contained 75 mM sodium thiocyanate, which favored the detectionof high avidity anticapsular antibodies as compared to low avidityantibodies (Raff et al. (1996) “Correlation between ELISA andbactericidal activity in infants and toddlers immunized with a MenC-CRMconjugate vaccine,” in Abstracts of the 36th Interscience Conference onAntimicrobial Agents and Chemotherapy, page 158 (Abstract)). The IgGmeningococcal C anticapsular antibody concentrations in test samples arereported in arbitrary units per ml, compared to that present in aninternal reference serum pool prepared from serum samples fromvaccinated healthy adults. For comparison, the meningococcal referenceserum pool CDC1992 (provided by George Carlone, Centers for DiseaseControl and Prevention, Atlanta, Georgia) (Gheesling et al. (1994) J.Clin. Microbiol. 32:1475-1482) contained 19.1 units/ml of themeningococcal C anticapsular antibody as measured by this modifiedassay.

Complement-mediated bactericidal antibody to Neisseria meningitidisgroup C was assayed as previously described, (Granoff et al. (1997)Infect. Immun. 65:1710-1715; and Mandrell et al. (1995) J. Infect. Dis.172:1279-1289) with the following modifications. The group C testorganism (N. meningitidis group C, strain 60E, obtained from Dr. W.Zollinger, Walter Reed Institute for Medical Research, Washington, D.C.)was grown for approximately 2 hours in Mulleur Hinton (MH) brothcontaining 0.25% glucose, which rendered the organism resistant tocomplement-mediated bacteriolysis by endogenous “natural” antibodies, ascompared to organisms grown in Mulleur Hinton without supplementalglucose. All test sera were heated at 56° C. for 30 mins to inactivateendogenous complement. The complement source for the bactericidal assaywas pooled sera obtained from three healthy adults who had no detectableanticapsular antibody to meningococcal C, and whose sera lackedintrinsic bactericidal activity when tested at 40 percent. In thebactericidal assay, this complement source was used at 20 percent in thefinal reaction mixture, along with serial 2-fold dilutions of test serabeginning at a 1:8 dilution (12.5 percent in the final reaction) andGey's buffer (instead of barbital buffer as previously described)(Mandrell, supra). Serum bactericidal titers were defined as thedilution of test sera resulting in a 50% decrease in colony formingunits per ml after 60 minutes incubation of bacteria in the reactionmixture, compared to control bacteria at time 0. Note that the titersreported with this assay tend to be lower than those described inprevious studies (Anderson, supra and Maslanka et al. (1997) Clin.Diagn. Lab. Immunol. 4:156-167). The principal reasons are: (1) the useof a test organism grown with supplemental glucose; (2) the use of Gey'sbuffer instead of barbital buffer (Mandrell, supra) and (3) the use ofhuman as opposed to rabbit complement, since in previous studies rabbitcomplement was shown to amplify greatly bactericidal activity of humanantibodies (Mandrell, supra and Zollinger et al. (1983) Infect. Immun.40:257-264). Human complement also was chosen for the present studybecause the data demonstrating that serum bactericidal antibodycorrelated with protection of humans against invasive meningococcaldisease were derived from studies that used human complement(Goldschneider et al. (1969) J. Exp. Med. 129:1307-1326).

Statistical analysis: Antibody concentrations were transformed (log₁₀).For these calculations, bactericidal titers less than 1:8 were assignedas 1:4, and IgG antibody concentrations less than 0.4 units/ml wereassigned a value of 0.2 units/ml. Geometric means and 95% confidenceintervals were computed by using the log transformed means and standarderrors were computed from a one-way analysis of variance (ANOVA) model.Differences between each pair of groups with respect to geometric meanswere tested by using the P values from the ANOVA model.

B. Results

Clinical tolerability: Vaccination with {fraction (1/50)} of the usualdose of MENOMUNE™ was well tolerated irrespective of previousmeningococcal vaccination status. During the 28 days of follow-up, therewere no clinically significant local reactions at the injection site orsystemic reactions, such as fever, rash, or myalgia, in any of the 34subjects.

Antibody response: FIG. 1 shows the geometric mean IgG anticapsularantibody responses of each group to the booster vaccination. Prior tothe vaccination, there were no significant differences between thegeometric mean antibody concentrations of the three groups (0.30, 0.78and 0.73 units/ml, for groups 1, 2, and 3, respectively). At 3 daysafter vaccination, there was no evidence of a significant increase inserum IgG antibody concentrations in any of the groups, when compared tothe respective antibody concentrations present in pre-vaccination sera.However, by 7 days, subjects in group 2, who previously had received theconjugate vaccine, and subjects in group 3, who were vaccinated for thefirst time, showed significant IgG responses, compared to theirrespective IgG serum antibody concentrations present at time 0 (p<0.05for each group). In contrast, the adults in group 1, who had received afull dose of licensed meningococcal A and C polysaccharide vaccine 4years earlier, showed no evidence of an IgG response at 7 days(geometric mean IgG antibody concentration of 0.38 units/ml at 7 daysvs. 0.42 units/ml at time 0). Similarly, there was no significantincrease in geometric mean antibody concentration in this group whenmeasured at 28 days (0.68 units/ml, P>0.5). At 28 days, only one subjectin group 1 showed a ≧4-fold increase in IgG antibody concentration, andthe respective geometric mean IgG antibody responses were 6- to 10-foldlower than those of groups 2 or 3 (p<0.003 at 7 days; and p<0.01 at 28days).

Table 1, below, summarizes the bactericidal antibody responses of thethree groups as measured in serum samples obtained at time 0, and 7 and28 days after the booster vaccination (bactericidal assays were notperformed in the sera obtained at 3 days). Prior to the boostervaccination, all five subjects in group 1 had titers less than 1:8(undetectable), and the majority of adults in groups 2 and 3 also hadundetectable bactericidal titers (Table 1). Following vaccination, thegeometric mean bactericidal antibody responses at 7 and 28 daysparalleled the respective IgG antibody responses. Specifically, at 7days there was no evidence of an increase in bactericidal antibodytiters in group 1, compared to the respective pre-vaccination titers,and the GMT of group I at 7 days was 10- to 25-fold lower than therespective GMTs of groups 2 or 3 (p<0.001). At 28 days, similarrespective differences were present (P<0.006). Further, at 28 days, theproportion of subjects with bactericidal titers ≦1:8 was only 20% ingroup 1, vs. 100% for group 2 (P=0.01), vs. 64% for Group 3 (P=0.11).

TABLE 1 Complement-Mediated Bactericidal Antibody Responses of Adults toa Meningococcal Polysaccharide Booster Immunization* Geometric Mean(Reciprocal Titer) Meningococcal No. ±95% Confidence Interval Percentwith Titer ≧ 1:8 Group Priming Vaccine Tested Pre- 7 Days Post 28 DaysPost Pre- 7 Days Post 28 Days Post 1 Polysaccharide  5 4.0 4.9 8.5 0 2020 (4-4) (2.8-8.6) (1.1-68)  2 Conjugate 18 9.3 136 200 22 100 100 (4-21)  (69-268) (108-371) 3 Unvaccinated 11 9.2 43 88 36 64 73(3.4-25)   (11-174)  (17-463) *For the booster injection, all subjectswere given 1/50th of the usual dose of Menomune (1 μg of eachpolysaccharide, IM). Statistical Analysis: The group previously givenMenomune showed no significant antibody response to the booster.Comparing GMTs of the 3 groups at each time point (Pre, p = 0.5; 7 days,p < 0.001; and 28 days, p = 0.005). # Pair-wise comparisons between GMTsof group previously vaccinated with Menomune vs. unvaccinated: 7 days, p= 0.01; and 28 days p = 0.02). Pair-wise comparisons between GMTs ofgroup previously vaccinated with conjugate # vaccine vs. unvaccinated: 7days, p = 0.06; and 28 days, p = 0.24. Pair-wise comparisons of percentwith titers ≧ 1:8 between group previously vaccinated with conjugate vs.unvaccinated: 7 days, p < 0.02; and 28 days, p < 0.05 (by Chi squareanalysis).

The principle finding of this study is that four years afterimmunization with meningococcal polysaccharide vaccine, healthy adultsshow much lower anti-meningococcal C serum antibody responses to abooster injection with {fraction (1/50)}th of the usual dose ofmeningococcal polysaccharide vaccine than adults vaccinated for thefirst time. In contrast, the magnitude of the booster responses ofadults previously vaccinated with an investigational meningococcalconjugate vaccine was similar or higher than that of the adultsvaccinated for the first time. These data are consistent with inductionof immunologic tolerance to meningococcal C polysaccharide by the priorvaccination with the licensed polysaccharide vaccine, but not by theinvestigational conjugate vaccine. The conclusion that the initialunconjugated polysaccharide vaccination in adults induced immunologictolerance is based on the booster responses of 5 subjects, wherein themagnitude of the impairment found was very large (10-fold), and thusunlikely to have resulted from chance alone (P≦0.01).

One contributing factor in the mechanism for the above-describedinduction of immunologic tolerance may be the relatively high dose ofpolysaccharide used in the licensed meningococcal vaccine (50 μg). Thatthis dose may be excessive is suggested by the excellent immunogenicityof a 1 μg dose in the control adults immunized in the present study forthe first time (FIG. 1 and Table 1). Also, in a previous study ininfants, impaired booster antibody responses to meningococcal Cpolysaccharide were observed only after vaccination with 25 μg or 100 μgof vaccine, but not after a 10 μg dose (Goldschneider et al. (1973) J.Infect. Dis. 128:769-776). In mice, large doses of polysaccharideantigens also have been found to induce immunologic tolerance, whereaslower doses are immunogenic and do not induce a refractory state torevaccination (reviewed in Halliday, supra) Accordingly, novel methodsfor boosting anti-meningococcal immune responses in adults, and uses offirst and second meningococcal vaccine compositions in the preparationof medicaments are disclosed. Although preferred embodiments of thesubject invention have been described in some detail, it is understoodthat obvious variations can be made without departing from the spiritand the scope of the invention as defined by the appended claims.

What is claimed is:
 1. A method for boosting in an adult subject animmune response against meningococcal C capsular antigen, said methodcomprising: (a) administering a first vaccine composition to said adultsubject in order to elicit an immune response against a meningococcalspecies, wherein said first vaccine composition is a meningococcalglycoconjugate vaccine composition that comprises meningococcaloligosaccharides from serogroups A and C, wherein the oligosaccharidesare conjugated to a carrier molecule, and further wherein the firstcomposition is administered in an amount sufficient to elicit ananti-meningococcal immune response; and (b) administering a secondvaccine composition to said adult subject in order to boost theanti-meningococcal response, wherein said second vaccine compositioncomprises capsular polysaccharides from serogroups A, C, Y and W135, andis administered to the subject about three to four years after the firstvaccine composition is administered.
 2. The method of claim 1, whereinthe oligosaccharides of the first vaccine composition are conjugated toa protein carrier.
 3. The method of claim 2, wherein the protein carrieris CRM₁₉₇.
 4. The method of claim 1, wherein the second vaccinecomposition comprises an unconjugated meningococcal polysaccharide. 5.The method of claim 4, wherein the second vaccine composition comprises1 μg of each polysaccharide.
 6. The method of claim 1, wherein thesecond vaccine composition is administered to the adult subject not lessthan three years after administration of the first vaccine composition.7. A method for producing an immune response against meningococcal Ccapsular antigen in an adult subject, said method comprising: (a)administering a first vaccine composition to said adult subject in orderto elicit an immune response against a meningococcal species, whereinsaid first vaccine composition is a meningococcal glycoconjugate vaccinecomposition that comprises meningococcal oligosaccharides fromserogroups A and C, wherein the oligosaccharides are conjugated toCRM₁₉₇, and further wherein the first composition is administered in anamount sufficient to elicit an anti-meningococcal immune response; and(b) administering a second vaccine composition to said adult subject inorder to boost the anti-meningococcal response, wherein said secondvaccine composition comprises capsular polysaccharides from serogroupsA, C, Y and W135 wherein said capsular polysaccharides are notconjugated to a carrier molecule, and further wherein said secondvaccine composition is administered to the subject about three to fouryears after the first vaccine is administered.
 8. The method of claim 7,wherein the second vaccine composition comprises 1 μg of eachpolysaccharide.